A fluorogenic substrate for the continuous assaying of aryl sulfatases

  • Ahmed V
  • Ispahany M
  • Ruttgaizer S
 et al. 
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Abstract

The most common fluorogenic substrate for assaying aryl sulfatases (ARSs) is 4-methylumbelliferyl sulfate (MUS). However, ARSs operate optimally at pH values that are less than the pKa(7.8) of the reaction product of MUS, 4-methylumbelliferone (4-MU). Thus, a major disadvantage of this assay is that it is usually run in a discontinuous mode due to the need for basification of the reaction mixture to achieve complete ionization of the phenolic products and maximum fluorescence. To circumvent this problem, 6,8-difluoro-4- methylumbelliferyl sulfate (DiFMUS) was prepared and examined as a substrate for ARSs. The product of the reaction is 6,8-difluoro-4-methylumbelliferone, a known coumarin with fluorescent properties equal to those of 4-MU, and has a pKaof 4.9. This allowed for the continuous assaying of human placental ARSs A, B, and C, which operate optimally between pH 5.0 and pH 7.0. Furthermore, DiFMUS exhibited a lower Km(up to 20-fold) for the ARSs than did MUS; for ARSA and ARSB, it exhibited a greater Vmaxthan did MUS. This substrate should have considerable utility for the continuous assay of ARS activity. © 2005 Elsevier Inc. All rights reserved.

Author-supplied keywords

  • 6,8-Difluoro-4- methylumbelliferyl sulfate
  • Aryl sulfatases
  • Continuous
  • Fluorescence assay

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Authors

  • Vanessa Ahmed

  • Mehdi Ispahany

  • Scott Ruttgaizer

  • Guy Guillemette

  • Scott D. Taylor

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